巴氏毕赤酵母SMD1168表达人溶菌酶的发酵条件

Fermentation Condition for the Expression of Human Lysozyme by Pichia pastoris SMD1168

研究用毕赤酵母SMD1168菌株(MutS,HIS4+)表达人溶菌酶(HLZ)的发酵条件。此菌株具有对胞外分泌的外源蛋白不降解、对反应器氧传递效率的限制不敏感的特性,有利于在常规反应器条件下的过程放大。在培养基中添加复合维生素,在流加甲醇溶液中添加山梨醇或甘露醇可维持细胞正常的生长和代谢,并表达高活性的HLZ。采用恒溶解氧的方式流加甘油溶液以提高细胞密度,在一段时间内使细胞处于碳源半饥饿状态后,使用恒速流加甲醇溶液诱导HLZ表达。HLZ的体积生成速率达到9.6mg/(L·h)左右,上清液中HLZ的含量约为1.6g/L,发酵周期为110h。

The process for the expression of human lysozyme (HLZ) by Pichia pastoris SMD1168 (MutS, HIS4+) was studied, and the volumetric rate of the HLZ production, about 9.6 mg/(L·h), and the HLZ in the supernatant, about 1.6 g/L, were achieved after 110 h of cultivation. The heterologous protein expressed and secreted in the broth would not be degraded by vacuolar proteases of yeast, and the growth of cells and the reactions of the methanol induction were insensitive to the limitation of oxygen transfer in commercial bioreactor, so this strain is suitable for production of engineered protein in industrial scale. The supplements of the complex vitamins in the medium and sorbitol or mannitol in the feed of methanol solution was necessary for the normal growth and metabolism of the cells to express HLZ with higher activities. The feed strategies for glycerol and methanol must be satisfied.